This project is aimed at studying cell tropism of HIV infectivity in vitro. Initially we developed a focal infectivity assay using plastic-adherent CD4-positive HeLa cells as targets for HIV infection in vitro. Subsequently this approach was used to study human glioma and squamous cell carcinoma cell lines expressing the same CD4 vector as the HeLa cells. Surprisingly these other cell lines could not be infected directly by HIV. This block in infectivity was found to be at the point of viral entry because the cells produced infectious HIV after transfection with viral DNA or infection with HIV pseudotyped by amphotropic murine leukemia virus. These results indicate that other cellular molecules in addition to CD4 are required for HIV fusion and entry into target cells. Additional work has been done using a new clone of CD4-positive HeLa cells to assay expression of infectious HIV in blood cells of seropositive human patients. The results indicated that HIV infectivity was expressed at very low levels, up to a maximum of 1 in 10,000 peripheral blood mononuclear cells. The new clone of HeLa cells was 300-fold more sensitive to infection than previous clones, and HIV isolates from 95% patients could be detected in these cells. However, virus from 5% of patients did not infect the CD4-positive HeLa cells. Current work on these isolates suggests that they are probably macrophage-tropic virus strains.